The benzoguinoid ansamycins have proven to be selectively cytotoxic in vitro to poorly differentiated solid tumors. Cytotoxicity does not correlate with alterations in protein tyrosine kinase activity or cellular phosphotyrosine content. Our focus in this project is now to determine the mechanism of selective cytotoxicity of these compounds. We have determined that all cytotoxic ansamycins have the property of binding to the heat shock protein hsp90 within 30 min of drug addition to cells. The sequelae of this protein binding are currently under investigation, but we can clearly show a disruptive effect on src activity, erbB2 kinase, and mutated p53. In screening a wide variety of ansamycin derivatives, we have determined that a very strong correlation exists between ability to bind hsp90 and cytotoxicity. Since hsp90 is involved in many intracellular functions, including glucocorticoid receptor function and raf kinase activity, the cytotoxic effects of the ansamycins probably involve disruption of specific hsp90/protein interactions which are critical to the proliferation/survival of certain cell types. In screening over 30 drugs of various structures (identified by the Drug Screening Program, DTP), we have found that only certain ansamycin derivatives are capable of interacting with hsp90. Another hsp90 family member, the glucose regulated protein GRP94, also binds geldanamycin. GRP94 forms a stable complex with erbB2 kinase which is disrupted by geldanamycin, and the disruption of this complex leads to destabilization of the erbB2 protein. Hsp90 also is required for the correct folding of certain forms of mutated p53. In this case, its interaction with p53 is transient.